Telomeres, the ends of linear chromosomes, are a challenge for DNA replication. In the absence of a specialized replication mechanism, DNA is lost from chromosome ends with each round of DNA replication. In most eukaryotes, the end replication problem is solved by telomerase, a telomere specific reverse transcriptase that extends the G-strand of telomeric DNA using its RNA component as a template. Telomerase action in S. cerevisiae in vivo requires at least five genes, EST1, EST2, EST3, TLC1 and CDC13. Deficiencies in any of these genes lead to progressive telomere shortening and eventually cell death. In addition, either of the checkpoint kinases Tel1p or Mec1p is required for telomerase-mediated telomere lengthening. Yeast telomerase is regulated by both the cell cycle and telomere length. The overall goal of the proposed research is to elucidate the molecular details of both types of regulation using a combination of genetic, biochemical, and cell biological approaches. Yeast telomerase lengthens telomeres only in late S/G2 phase, even though the catalytic subunit, Est2p, is telomere associated throughout most of the cell cycle. Est1p and Est3p are telomere associated only at the time of telomerase action, late S/G2 phase. In addition, telomerase acts preferentially at short telomeres, and Est1p, Est2p and Tel1p bind preferentially to short telomeres. We will determine if preferential binding of Tel1p to short telomeres is required for their preferential lengthening, examine how Tel1p is recruited to short telomeres, and determine if Mec1p binds short telomeres in tel1 cells. The effects of the negative regulators Rif1p and Rif2p on telomerase recruitment will be examined. We will test if Rif1p or Rif2p binding is depleted at telomeres that are shortened from their ends (rather than from internal deletion) using a new assay that combines chromatin immuno-precipitation (ChIP) with sequence analysis of individual telomeres (NRA, nucleotide resolution assay). Using a new system to generate a single long telomere, we will determine if Rif binding is enriched at long telomeres. If differential binding by telomere length is seen, we will determine the consequences of this behavior for the telomere association of Tel1p and telomerase. Rif1p has multiple candidate Tel1p phosphorylation sites: experiments to test if Rif1p is phosphorylated by Tel1p in a telomere length dependent manner will be conducted. If Rif1p is a Tel1p target, we will assess the consequences of its phosphorylation on telomerase recruitment. Our recent success in purifying Est1p, Est3p, and Cdc13p will be exploited to determine the properties of these proteins, such as their ability to bind DNA and RNA, and their effects on telomerase activity in vitro. These in vitro approaches will complement in vivo studies on Est1p, Est3p and Cdc13p, including use of NRA+ChIP to determine if these proteins are preferentially associated with those telomeres that are actually lengthened. We will also isolate gain of function est3 and est1 alleles to obtain insights into how these proteins function in the telomerase pathway and use cell biological approaches to test the hypothesis that telomere association with the periphery sequesters them from telomerase. Most human somatic cells do not express telomerase, and as a result their telomeres slowly shorten. Short telomeres can initiate genome instability which can lead to cancer or stem cell failure. Most human tumors, regardless of tumor type, express telomerase, and this expression probably contributes to their unconstrained growth. Many of the proteins involved in telomere length regulation in humans were first identified in yeast, which continues to serve as key model for understanding the human enzyme.